Method of producing porous spherical particles

ABSTRACT

The invention refers to a method of controlling the porosity of porous spherical particles produced from a polysaccharide dissolved in a solvent, in which it can be gelled. The polysaccharide solution is finely divided by mechanical means into spherical droplets which are allowed to pass through a humid atmosphere and transferred to a capturing medium while controlling the temperature and humidity of humid atmosphere.

[0001] The present invention refers to a method of producing porousspherical gelled particles. More precisely, the invention refers to amethod of controlling the porosity of porous spherical particlesproduced from a polysaccharide dissolved in a solvent, in which it canbe gelled, said polysaccharide solution being finely divided bymechanical means into spherical droplets which are allowed to passthrough a humid atmosphere and transferred to a capturing medium.

[0002] Gel filtration is a commonly used chromatographic separationmethod in which molecules are separated with respect to their size.Small molecules diffuse into porous particles while large molecules passon the outside thereof. Spherical particles intended for the separationof macromolecules by means of such a chromatography are produced from agelled and/or polymerized material. Of course, a good separation doesnot only depend on the size and the size distribution of the particlesbut also on the extent of the porosity of the particles.

[0003] In this connection the porosity of porous particles refers to theapparent size exclusion dimensions of a porous matrix as described byHagel et al. (“Apparent Pore-Size Distributions of ChromatographyMedia”, J. Chromatogr. 743(1):33-42, 1996).

[0004] Porous matrices, which are used for gel filtration, can after oneor several modifications with advantage also be used for othertechnologies including chromatographic separations. In thesetechnologies, e.g. ion exchange chromatography (IEX), hydrophobicinteraction chromatography (HIC), affinity chromatography, reversedphase chromatography etc, various interactions with direct and/orsubstituted ligand(s) on the gel are utilized, which are also highlydependent on the porosity. Such matrices can also be used for controlledrelease in connection with drug delivery.

[0005] Normally, these particles are produced by means of dispersionprocedures which are based on a vigorous mechanical mixing of thegenerally water soluble substance which is to be dispersed in adispersing medium usually comprising an organic solvent such as toluene.The processing of the spherical particles obtained from the dispersingprocedure takes place in several steps accompanied by a thorough rinsingin order to remove the solvent. This conventional method of producingthe particle shaped material for chromatography is thus energy demandingas well as expensive and, in addition, the productivity is low andproduced batchwise.

[0006] One way of avoiding organic solvents when producing suchparticles would be to use water as a solvent in the manufacturingprocedure. For example, a method for producing spherical alginateparticles is shown in the publication WO 93/02785, the alginate solutionbeing finely divided into droplets which are allowed to fall freelythrough the air down into an ionic solution in which they are hardened.

[0007] In experiments of producing particles from water solublepolysaccharides attempts have been made to allow droplets of apolysaccharide solution to gel directly into particles in water of 0-25°C. The results obtained in this connection have, however, not beensatisfactory with reference to such problems as shape, gel concentrationand surface face properties.

[0008] It is thus important that droplets of uniform size are produced.In WO 95/20620 spherical gel particles for chromatographic use areproduced by mechanical disintegration of a gelling liquid polymer andtransport of the particles through a gas to a solvent. In this way,particles are produced without the use of the solvents needed for anemulsion polymerization process, and the process yields particles ofnarrower size distribution.

[0009] The problem of water solubility during the collection in water ofspherical particles produced from agarose has been avoided in U.S. Pat.No. 5,053,332 by allowing the droplets—until they are gelled—to fallthrough and remain suspended in an upward flowing cooling gas which isinert to agarose. Such a procedure has a desiccating effect on theparticles.

[0010] The purpose of the invention is to avoid the above-mentioneddrawbacks of the known technique by producing an improved porousspherical material which preferably is intended for chromatography.

[0011] For water soluble polymeric materials the high Is demands thatorganic solvents must not be present in the material intended forchromatography are eliminated by the invention. Furthermore, newprocesses are obtained by the invention, in which organic solvents canbe avoided, an aspect which is important from an environmental point ofview.

[0012] More specifically, the invention refers to a method of producingporous spherical particles from a polysaccharide in solution, which canbe gelled and/or polymerized, the polysaccharide solution being finelydivided by mechanical means into spherical droplets. The droplets areallowed to pass through a controlled atmosphere, and the finely dividedmaterial formed is then allowed to pass (e.g. fall down) into acapturing medium. In this way, spherical particles having a narrowparticle size distribution can be continuously produced from apolysaccharide.

[0013] Such particles can for example be achieved with the devicedescribed in WO 88/07414. With this system, droplets having a diameterof 20-500 μm can be produced.

[0014] The method according to the invention is based on the fact thatwhen a polysaccharide, which can be gelled, is formed into a droplet,the droplet under certain circumstances is gelled into a threedimensional lattice enclosing a solvent in such a way that a pseudosolidmaterial is formed. Thus, the molecular structure in the droplet formedhas—from the very beginning—defined the maximal porosity of a futureparticle. The structure determining the maximal porosity is initiallycreated in a droplet and the final pore formation is the result of acombination with a subsequent partly irreversible evaporation anddesiccation process.

[0015] Accordingly, it is very important that the pore formation in thedroplets takes place under controlled conditions during the gelling ofthe polysaccharide. If the evaporation or the desiccation is tooextensive, the surface of the future particles will dry and an irregularsurface will be obtained the porosity at the same time beingconsiderably affected. The risk of an uncontrolled desiccation increaseswhen a smaller particle size is contemplated.

[0016] The controlled atmosphere is according to the invention a humidatmosphere, the temperature and humidity of which is controlled. Porousspherical particles of retained or increased porosity are obtained aftera conveyance through an atmosphere, which thus consists of a varyingmixture of air and water in gaseous phase or water vapor only. Theatmosphere used is also controlled by its temperature. A temperaturegradient will inevitably be obtained, but it can be controlled.Preferably, the temperature gradient is controlled to be as smooth aspossible from the site of droplet formation to the capturing medium.

[0017] After this controlled conveyance through a humid atmosphere, thepolysaccharide droplets are captured. In the capturing medium, thegelling of the droplets/particles is completed. Preferably, thecapturing medium comprises water, but it can also be an organic solvente.g. toluene, when a low surface tension is of importance for themanufacturing of completely spherical particles.

[0018] The porous spherical particles formed can then be separated bymeans of filtration or sedimentation.

[0019] The droplets from a particle generating apparatus contain apolysaccharide solution, which can be gelled, and the gelling may or maynot be continues during the conveyance through the controlledatmosphere. Thus, the temperature of the humid atmosphere is higher thanthe gelling temperature of the polysaccharide. The controlled atmospherecan be of the same temperature as the environment, for example roomtemperature, but higher as well as lower temperatures may be used independence of the application contemplated. Accordingly, thepolymerization of a material, which can be gelled, can also becontrolled to take place during the conveyance through the controlledatmosphere. The residence time in this atmosphere is usually less thanabout 30 seconds, but can of course be varied.

[0020] This procedure results in that the surface porosity of the newlyformed particles is maintained throughout the gelling stage. In thisconnection, a desiccation of the particle surface is avoided during thegelling, which would result in an extensive reduction of the porosity.

[0021] In principle, the polysaccharide used for exercising the methodaccording to the invention can be any naturally occurringpolysaccharide. Preferably, the polysaccharide is agarose, agar, starch,or alginate.

[0022] The polysaccharide is dissolved in a solvent comprising water. Ifthe gelling polysaccharide is agarose, it is made as water solution of2-14 weight %. In this case, the gelling process takes place at a highrelative humidity and at a high temperature. Preferably, the cooling ofthe droplet takes place in a water vapor (100% relative humidity). Thedroplets obtained are conveyed to a capturing medium through anatmosphere controlled with respect to humidity and temperature, whichpreferably consists of a mixture of air and water vapor, alternativelywater vapor only. During the conveyance, the droplets are subjected to atemperature gradient, preferably between 100° C. and 20° C., thesteepness of which can be controlled. The final gelling can also beachieved in the capturing medium.

[0023] The polysaccharide gel solution as well as the aqueous capturingmedium can advantageously also contain additives affecting the gelling,such as salts etc. Since the polysaccharide is substantially soluble inwater, a complete gelling at low temperature can be effected in theaqueous capturing medium. The capturing medium can also contain asurface tension reducing agent in order to facilitate the transportthrough the surface of the medium.

EXAMPLES

[0024] The following non-limiting examples will now be given in order tofurther describe the invention.

Example 1.

[0025] Agarose was dissolved to 4 weight % in boiling water, and thesolution was then allowed to cool to 90° C. while stirring. At thistemperature the agarose solution obtained was finely divided intospherical droplets of 100μm±25 μm by means of the device described in WO88/07414. Then, for about 30 seconds the spherical droplets obtainedwere allowed to fall through a zone of water vapor/air of a highmoisture content (50-100%) and temperature (20-100° C.). The particleswere then allowed to fall down into a tank containing water of roomtemperature, the particles being completely gelled therein. Thepreparation of one separate batch for chromatography results in aparticle size distribution of more than 80% of the material within ±25%of the mean particle size.

Example 2.

[0026] Agarose particles were produced by disperging 6.6 g of agarosepowder to 100 ml of water having a temperature of about 30° C. Theagarose powder suspension was then completely dissolved with stirring ina microwave oven at about 96° C.

[0027] The gel solution obtained was sprayed by means of a rotating diskinto individual droplets, as shown in WO 88/07414. The droplets formedwere caught in water, which resulted particles having a diameter of 100μm±10% and a dry weight of about 6% (w/v).

[0028] Before being captured, the droplets formed by the rotating diskwere allowed to pass through a controlled atmosphere according to theinvention. This was accomplished by arranging a dome over the spinningdisk and controlling temperature and humidity underneath to be 50° C.and 100%, respectively.

[0029] As a comparison, the droplets formed were allowed to pass normalroom atmospheric conditions, i.e. a temperature of 20° C. and a humidityof 55%.

[0030] The porosities of the particles formed were compared afterpassage through the different atmospheres and transfer to a capturingmedium. This was performed in column (Ø=10×h=300 mm) which wasequilibrated and eluted with 25 mM Tris-HCl, pH 7.0, and 100 mM KCl at arate of 15 ml/h. The column was loaded with different substances ofknown molecular weights. Any appearance of a substance after the voidvolume was noted (Yes), which indicated diffusion of he substance intothe porous spherical particles. The results are given in Table 1 below.TABLE 1 Substance Mw (D) Comparison Invention Thyroglob. 669 000 0 YesFerritin 440 000 0 Yes Catalase 232 000 Yes Yes Aldolase 158 000 Yes Yes

[0031] The results show that particles produced according to theinvention have porosities that allow diffusion of substances of verylarge molecular weights, up to more than 650 000 Dalton. Particles undernormal atmospheric conditions resulted in less porous particles. Theyonly allow diffusion of considerably smaller substances, i.e. less than250 000 Dalton. Consequently, particles produced according to theinvention demonstrate increased porosity.

1. Method of controlling the porosity of porous spherical particlesproduced from a polysaccharide dissolved in a solvent comprising water,the polysaccharide solution being finely divided by mechanical meansinto spherical droplets and transferred to a capturing medium,characterized in that said droplets are conveyed through a humidatmosphere, the temperature and, humidity of which being controlled. 2.Method as in claim 1, characterized in that said temperature and/or saidhumidity is set above ambient temperature and/or humidity, respectively,said porosity being retained or increased.
 3. Method as in claim 2,characterized in that the temperature of said capturing medium isbrought below that of the gelling temperature of the polysaccharide. 4.Method as in claim 1, characterized in that said capturing mediumcomprises water.
 5. Method as in any of claims 1 to 4, characterized inthat said polysaccharide is selected from the group comprising agarose,cellulose, starch, and alginate.
 6. Method as in claim 5, characterizedin that when said polysaccharide is agarose, it is dissolved as a 2-14weight % water solution.